Gene activation requires the tight coordination of regulatory factors at the site of transcription. We propose to study the recruitment dynamics of transcriptional regulatory factors to a chromatinized transcription site in single living cells in order to produce a high-resolution temporal and spatial portrait of transcriptional activation. We will use shRNA depletion to evaluate the requirement of known regulatory factors on chromatin decondensation, pre-initiation complex assembly and RNA synthesis. We will also determine the kinetic and spatial dynamics of post-translational modifications (PTM) and PTM binding proteins during transcription activation and mitosis. In order to expand the use of the live cell imaging methodology, we also propose to use p53 as a model transcriptional activator and to interrogate the timing of its activation kinetics. These studies are significant because they cannot be done using other existing techniques that average effects in cell populations. Additionally, they will provide a portrait of unperturbed gene regulation in single cells. A comprehensive understanding of transcriptional regulation is important for the development of new strategies to cure human diseases. PUBLIC HEALTH RELEVANCE: Numerous diseases, including cancer, are caused by aberrations in transcriptional regulation. Gene activation requires the tight coordination of regulatory factors at the site of transcription. We propose to study the recruitment dynamics of these factors at a transcription site in single living cells in order to produce a high- resolution map of their recruitment order. A comprehensive understanding of transcriptional regulation is important for the development of new strategies to cure human diseases.